Our overall goal is to define the bacterial stress response in Pseudomonas aeruginosa that activates genes for the production of the capsular polysaccharide alginate, an important virulence factor in pathogenesis and especially in pulmonary disease. P. aeruginosa is one of the most deadly opportunistic pathogens and is notorious for chronic pulmonary inflammation in cystic fibrosis (CF) and COPD patients. For 27 years, this NIH-sponsored program has been focused on understanding the complex regulatory machinery associated with alginate gene activation. Our recent studies revealed that alternative ECF sigma factor, sigma-22, is not only the main activator of alginate gene expression, but also the key regulator of an envelope / cell wall stress response system. We discovered that exposure of the bacteria to antibiotics that interfere with cell wall synthesis causes sigma-22 activation by an intra-membrane regulated proteolysis (RIP) mechanism. Using microarrays, we identified 293 protein gene products under positive control by sigma-22 and also several putative small RNAs (sRNA) that are likely to have important regulatory functions within the sigma-22 regulon. AlgB is two-component regulator of alginate gene activation, and we have discovered a phospho-mimic mutant of AlgB that can activate alginate biosynthesis by using sigma-54, which suggests an alternate pathway for expression of the algD operon for alginate biosynthesis. We have also obtained evidence that the large 5' upstream untranslated region (5'UTR) in the algD operon's mRNA modulates its expression. The overall goal of the next phase of these studies is to examine the hypothesis that important regulatory RNA mechanisms, which respond to envelope stress, lead to alginate gene activation. We will pursue 3 aims that are directed at understanding this global stress response that activates alginate genes: 1. Define the regulatory sRNAs induced by sigma-22 activation, 2. Characterize the AlgB and sigma-54 dependent alternative pathway for alginate gene expression, and 3. Define the role of the large upstream untranslated region (5'UTR) of the alginate operon in the regulation of alginate gene expression.